Innate immune response of channel catfish Ictalurus punctatus mannose-binding lectin to channel catfish virus (CCV).

The channel catfish virus (CCV) is a pathogenic herpesvirus that infects channel catfish Ictalurus punctatus in pond aquaculture in the southeastern USA. Mannose-binding lectin (MBL), an innate immune protein, could play an important role in the innate response of channel catfish by binding to CCV. Cell cultures of CCV were grown in channel catfish ovary cells (CCOC). A dot-immunoblot enzyme-linked immunosorbent assay was done to determine the binding ability of 5 mo old channel catfish serum MBL (26.2 µg ml-1) to CCOC infected with CCV. Two separate nitrocellulose membrane blotting techniques were done using uninfected and infected CCOC. The uninfected CCOC decreased by 29.3 and 33.4% in their binding of channel catfish MBL when compared with infected CCOC using the 2 membrane procedures. The combined average binding ability of channel catfish MBL towards infected CCOC was therefore 31.4% greater when comparing the infected and uninfected CCOC. Normalization equation values of MBL for the 5 mo old catfish were compared for the 2 membrane binding procedures. The 2 normalization values were very close (142 and 150) in binding ability of MBL to the infected CCOC. The 5 mo catfish serum had twice the concentration of MBL (26.2 µg ml-1) compared to 7 mo catfish serum (13.2 µg ml-1), and the binding percentage of 5 mo serum was 2.4 times greater in infected than in uninfected cells. This demonstrates that the binding of channel catfish serum MBL to CCV is concentration dependent and is related to serum concentrations of MBL.

Coordinated regulation of Rel expression by MAP3K4, CBP, and HDAC6 controls phenotypic switching.

Coordinated gene expression is required for phenotypic switching between epithelial and mesenchymal phenotypes during normal development and in disease states. Trophoblast stem (TS) cells undergo epithelial-mesenchymal transition (EMT) during implantation and placentation. Mechanisms coordinating gene expression during these processes are poorly understood. We have previously demonstrated that MAP3K4-regulated chromatin modifiers CBP and HDAC6 each regulate thousands of genes during EMT in TS cells. Here we show that CBP and HDAC6 coordinate expression of only 183 genes predicted to be critical regulators of phenotypic switching. The highest-ranking co-regulated gene is the NF-κB family member Rel. Although NF-κB is primarily regulated post-transcriptionally, CBP and HDAC6 control Rel transcript levels by binding Rel regulatory regions and controlling histone acetylation. REL re-expression in mesenchymal-like TS cells induces a mesenchymal-epithelial transition. Importantly, REL forms a feedback loop, blocking HDAC6 expression and nuclear localization. Together, our work defines a developmental program coordinating phenotypic switching.

GALNT3 Maintains the Epithelial State in Trophoblast Stem Cells.

O-GalNAc glycosylation is initiated in the Golgi by glycosyltransferases called GALNTs. Proteomic screens identified >600 O-GalNAc-modified proteins, but the biological relevance of these modifications has been difficult to determine. We have discovered a conserved function for GALNT3 in trophoblast stem (TS) cells, blastocyst trophectoderm, and human mammary epithelial cells (HMECs). The loss of GALNT3 expression in these systems reduces O-GalNAc glycosylation and induces epithelial-mesenchymal transition. Furthermore, Galnt3 expression is reduced in aggressive, mesenchymal claudin-low breast cancer cells. We show that GALNT3 expression controls the O-GalNAc glycosylation of multiple proteins, including E-cadherin in both TS cells and HMECs. The loss of GALNT3 results in the intracellular retention of E-cadherin in the Golgi. Significantly, re-expression of GALNT3 in TS cells increases O-GalNAc glycosylation and restores the epithelial state. Together, these data demonstrate the critical biological role of GALNT3 O-GalNAc glycosylation to promote the epithelial phenotype in TS cells, blastocyst trophectoderm, and HMECs.

MAP3K4 Controls the Chromatin Modifier HDAC6 during Trophoblast Stem Cell Epithelial-to-Mesenchymal Transition.

The first epithelial-to-mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TSKI4 cells) induces an intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships among MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TSKI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TSKI4 cells restores epithelial features, including cell-cell adhesion and barrier formation. These data define a role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes.